The recognition that one out of 4 breast cancers exhibits elevated levels of the epidermal growth factor receptor type 2 (HER2) is one of the most significant recent discoveries in breast cancer research. HER2 is associated with advanced disease and typifies cancers that are fast growing, highly metastatic, and resistant to treatment. As such, patients with HER2-positive breast cancers have a worse prognosis compared to patients with HER2-negative breast cancers. Despite recent advances in treatments, the outcome for HER2-positive breast cancer patients with advanced disease is death. Several reasons have been proposed for this. One reason is insensitivity or resistance to treatment resulting from the complex nature of the disease. The second reason is toxicity associated with the non-specific nature of the treatments themselves. Therefore, there is an increasing need for developing safer, more effective treatments for HER2-positive breast tumors. We propose to develop a novel reagent that is specific and circumvents many of the adaptive responses of HER2-positive breast cancers that lead to resistance. These properties will be achieved by engineering a multifunctional inhibitor composed of a targeting moiety (an RNA aptamer that binds to HER2) and a therapeutic moiety (a cytotoxic siRNA to breast cancer specific pro-survival factors). Upon binding to HER2 on the cell-surface, the reagent will deliver its therapeutic cargo into the cells resulting in cancer cell death. In addition to inhibiting pro-survival factors, this reagent has the potential to antagonize the activity of HER2 itself by promoting its degradation (combination therapy). During the five-year project we propose to validate this approach in proof-of-concept studies using rodent models of breast cancer. The knowledge derived from this work will be used to develop a human-specific therapeutic modality with emphasis on safety and therapeutic efficacy. Our specific aims are to (1) apply cutting-edge RNA technologies to inhibit pro-survival factors in HER2-positive breast cancer cells, (2) determine efficacy and safety of this reagent in rodent models of breast cancer, and (3) develop human-specific reagents and evaluate efficacy in human breast cancer cells and in mouse models of human breast cancer. At the completion of these studies, it is our expectation that we will have identified reagents for targeting HER2-positive breast tumors (as well as other HER2-expressing cancers). In addition, this work will develop general principles that can be applied towards the establishment of a platform technology for generating targeted gene silencing therapies against other cancers. The successful completion of this work is expected to provide clinicians as well as breast cancer patients, including patients with refractory disease, with improved treatment choices for fighting HER2-positive cancers of the breast.